The most common human aneuploidy is complete or partial trisomy of human chromosome 21 (HSA21), which results in Down Syndrome (DS). Trisomy 21 occurs at a frequency of 1 in 43 spontaneous abortions and 1 in 750 live births. Despite its high prevalence and intensive investigation, the molecular mechanisms leading to the development of phenotypic changes are poorly understood. The characteristics of DS include certain constant phenotypic characteristics such as mental retardation, craniofacial features, and immunodeficiency as well as inconstant characteristics such as congenital heart disease, placental vascular insufficiency, muscular hypotonia, and gastrointestinal malformations. The observation that a 1.5-fold increase in gene dosage can produce significant developmental effects suggest that the genes at fault might function synergistically. Recently, synergy between two genes DYRK1a and DSCR1 within the critical region of chromosome 21 was shown to reduce the nuclear occupancy of the NFATc proteins leading to the misregulation of genes critical to neural, skeletal, and immune development and function. NFATc proteins are both transcription factors and signaling proteins and are essential to many aspects of vertebrate development and morphogenesis. This signaling and transcriptional pathway is required for signaling by neurotrophins, netrins, FGF, VEGF, RANKL, the T lymphocyte receptor and Ca++ channels. Remarkably, the phenotypes of NFATc1, c2, c3 and c4 mutant mice reproduce severe forms of many or most of the characteristic features of Down syndrome. Feedback loops within the Calcineurin/NFAT pathway produce extraordinary sensitivity to a 1.5-fold increase of DYRK1a and DSCR1, which have been found to be over- expressed in tissues of patients with DS in virtually all published studies. Mathematical modeling predicts that increased DSCR1 and DYRK1a reduce nuclear occupancy of the NFATc proteins and lead to a failure to activate critical target genes and thereby to features of DS. Preliminary observations of mice with segmental trisomy and fetuses with trisomy have been consistent with this Trisomy Synergy Model. We propose to test this model of the pathogenesis of DS and if confirmed use this model to develop therapeutic leads to treat pathologic features of Down syndrome. Initially we will determine if samples from DS human fetuses, cord blood hematopoietic stem cells and T lymphocytes have the expected biochemical and genetic features of reduced NFAT activity and reduced target gene activation. We will formally test the role of DYRK1a and DSCR1 in murine models of DS by determining if normalizing the dosage of DYRK1a and DSCR1 rescues the DS-like characteristics in these mice. We will develop quantitative mathematical models of the effects of trisomy on the NFAT genetic circuit and expand these models to include interactions with other trisomic genes. This Trisomic Synergy Model will be useful for predicting potential sites of therapeutic intervention as well as predicting differences between gene dosage effects in mice and humans. Finally, we will develop small molecule screens for activators of NFAT-dependent transcription and test the ability of any molecules found to rescue the defects in DS T lymphocytes. At the conclusion of our studies we expect to have clearly defined the role of NFAT dysfunction in producing the phenotypic features of Down syndrome and to have discovered therapeutic leads for the treatment of non-developmental aspects of Down syndrome. Project Narrative: Down Syndrome is a common disorder caused by an additional copy of chromosome 21. Recent studies indicate that the extra chromosome imbalances a pathway controlling the development and function of the brain, skeleton, cardiovascular and immune systems. We will test this hypothesis and if correct find molecule that correct the balance and thereby develop new treatments for some of the disabling characteristics of Down Syndrome.